Prognosis in patients with hepatocellular carcinoma correlates to mutations of p53 and/or hMSH2 genes.

Association of gene alterations and prognosis has not fully been elucidated in hepatocellular carcinoma (HCC). To clarify the relationship between p53 and hMSH2 mutations and prognosis, we analysed these mutations in 83 HCC cases and assessed their association with various clinicopathological factors. The 3-year disease-free survival (DFS) or overall survival (OS) rates in HCC patients with p53 mutation and p53 wild/hMSH2 mutation significantly decreased compared with those without these mutations (14.3% and 37.5% versus 67.5% for DFS; 35.7% and 50.0% versus 96.4% for OS, respectively). In the multivariate analysis, categories by p53 and hMSH2 mutation status, and liver cirrhosis demonstrated statistical significances for DFS and OS. Moreover, the frequency of patients with p53 and/or hMSH2 mutations in intrahepatic metastasis (75.0%) was significantly higher than that in multicentric occurrence (14.3%). Thus, p53 and hMSH2 mutations will be useful for identifying subsets of HCC patients with poor prognosis.

A dose-dependent decrease in the fraction of cases harboring M6P/IGF2R mutations in hepatocellular carcinomas from the atomic bomb survivors.

The risk for hepatocellular carcinoma (HCC) development is significantly heightened in the atomic bomb survivors, but the mechanism is unclear. We have previously reported finding a radiation dose-dependent increase in HCCs with TP53 mutations from the survivors. We now show that, in the same HCC samples, the frequency of 3'-untranslated region (3'UTR) mutations in M6P/IGF2R, a candidate HCC tumor suppressor gene, decreases with dose (P = 0.0091), implying a radiation dose-dependent negative selection of cells harboring such mutations. The fact that they were in the 3'UTR implicates changes in transcript stability rather than in protein function as the mechanism. Moreover, these M6P/IGF2R 3'UTR mutations and the TP53 mutations detected previously were mutually exclusive in most of the tumors, suggesting two independent pathways to HCC development, with the TP53 pathway being more favored with increasing radiation dose than the M6P/IGF2R pathway. These results suggest that tumors attributable to radiation may be genotypically different from tumors of other etiologies and hence may provide a way of distinguishing radiation-induced cancers from "background" cancers--a shift from the current paradigm.

Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population.

Individuals who are homozygotes for mutations in DNA repair genes are at high risk for cancer. It is not well documented, however, if the heterozygous carriers of the mutation are also predisposed to cancer. To address the issue, xeroderma pigmentosum (XP) in Japan is an interesting candidate because of three major reasons: XP is an autosomal recessive disorder with an enormously elevated risk of skin cancer, the frequency of XP patients is higher in Japan than in other parts of the world, and more than half of Japanese XP patients are homozygous for the same founder mutation in the XPA gene. We screened archival blood samples from Japanese individuals who resided in Hiroshima or Nagasaki. A simple PCR-RFLP method was developed that is highly specific for detection of XPA heterozygotes carrying the founder mutation. We identified nine XPA heterozygotes among 1,020 individuals screened for a prevalence of 0.88%. This rate, if representative, implies that there are about 1 million carriers of the XPA founder mutation in the Japanese population. Thus, investigation of their cancer risk may be warranted.

Colorectal cancer with mutation in BRAF, KRAS, and wild-type with respect to both oncogenes showing different patterns of DNA methylation.

PURPOSE: BRAF mutations are common in sporadic colorectal cancers (CRCs) with a DNA mismatch repair (MMR) deficiency that results from promoter methylation of hMLH1, whereas KRAS mutations are common in MMR proficient CRCs associated with promoter methylation of MGMT. The aim of this study was to further investigate the link between genetic alterations in the RAS/RAF/ERK pathway and an underlying epigenetic disorder. PATIENTS AND METHODS: Activating mutations of BRAF and KRAS were identified and correlated with promoter methylation of 11 loci, including MINT1, MINT2, MINT31, CACNA1G, p16(INK4a), p14(ARF), COX2, DAPK, MGMT, and the two regions in hMLH1 in 468 CRCs and matched normal mucosa. RESULTS: BRAF V599E mutations were identified in 21 (9%) of 234 CRCs, and KRAS mutations were identified in 72 (31%) of 234 CRCs. Mutations in BRAF and KRAS were never found in the same tumor. CRCs with BRAF mutations showed high-level promoter methylation in multiple loci, with a mean number of methylated loci of 7.2 (95% CI, 6.6 to 7.9) among 11 loci examined (P < .0001). Tumors with KRAS mutations showed low-level promoter methylation, and CRCs with neither mutation showed a weak association with promoter methylation, with an average number of methylated loci of 1.8 (95% CI, 1.5 to 2.1) and 1.0 (95% CI, 0.79 to 1.3), respectively. CONCLUSION: In CRC, the methylation status of multiple promoters can be predicted through knowledge of BRAF and, to a lesser extent, KRAS activating mutations, indicating that these mutations are closely associated with different patterns of DNA hypermethylation. These changes may be important events in colorectal tumorigenesis.

Anisomycin downregulates gap-junctional intercellular communication via the p38 MAP-kinase pathway.

Phosphorylation of connexin 43 (Cx43) molecules (e.g. by extracellular signal-regulated kinase) leads to reductions in gap-junctional intercellular communication (GJIC). GJIC levels also appear to be lower in the presence of p38 mitogen-activated protein (MAP) kinase, for unknown reasons. In this study, we used assays of the recovery of fluorescence by photobleached WB-F344 cells to demonstrate that GJIC levels are decreased by anisomycin [a protein synthesis inhibitor as well as an activator of p38 MAP kinase and c-Jun N-terminal kinases (JNK)] as a result of time-dependent depletion of the phosphorylated forms of Cx43. Using immunohistochemistry, we also detected far less of the Cx43 proteins at cell borders. These findings agree with the photobleaching assay results. Moreover, prior treatment with SB203580 (a specific inhibitor of p38 MAP kinase) appeared to be effective in preventing the loss of phosphorylated forms of Cx43 and the loss of Cx43 proteins at cell borders. Total protein labelling with [(35)S]-methionine and [(32)P]-orthophosphates labelling of Cx43 showed that anisomycin enhanced the phosphorylation level of Cx43 along with inhibition of protein synthesis. SB203580 prevented the former but not the latter. The effect of anisomycin on GJIC was not dependent on the inhibition of protein synthesis because the addition of SB203580 completely maintained the level of GJIC without restoring protein synthesis. The Cx43 phosphorylation level increased by anisomycin treatment, whereas the amount of phosphorylated forms of Cx43 decreased, suggesting that activation of Cx43 phosphorylation might lead to the loss of Cx43. These results suggest that activation of p38 MAP kinase leads to reduction in the levels of phosphorylated forms of Cx43, possibly owing to accelerated degradation, and that these losses might be responsible for the reduction in numbers of gap junctions and in GJIC.

Allelic loss of a common microsatellite marker MYCL1: a useful prognostic factor of poor outcomes in colorectal cancer.

PURPOSE: Allelic loss involving chromosome arms 5q, 8p, 17p, and 18q is commonly detected in colorectal cancer (CRC). The short arm of chromosome 1 is also frequently affected in a whole range of cancer types, including CRC. Our aim in the present study was to determine whether allelic losses on 1p were likely to be of much value in predicting the prognosis of CRC cases. EXPERIMENTAL DESIGN: Genomic DNA was prepared from tumor and corresponding normal tissue specimens from 90 patients who had undergone curative resection for CRC. Loss of heterozygosity (LOH) on chromosome arms 1p, 2p, 5q, 7q, 8p, 17p, 17q, and 18q was examined using 14 microsatellite markers, and possible correlations between LOH and clinicopathological factors (including tumor recurrence and patient survival) were investigated. LOH at the MYCL1 microsatellite marker at 1p34 was detected in 12 of 74 (16.2%) patients who were informative for this marker. RESULTS: After controlling for tumor stage and gender and excluding findings for patients with remote metastasis, we found that patients who were positive for LOH at MYCL1 were 31 times more likely to experience recurrence than those who were negative for LOH at this locus (95% confidence intervals, 2.27- infinity; P = 0.04). There were indications of a similar tendency for LOH at the 14-3-3-sigma-TG microsatellite marker at 1p35, but we could find no evidence of a significant association between LOH at this site and tumor recurrence or patient survival. We were also unable to detect significant association between LOH at the various sites on 2p, 5q, 7q, 8p, 17p, 17q, and 18q and either tumor recurrence or patient survival. CONCLUSIONS: CRC patients whose tumors exhibited LOH at MYCL1 at chromosome 1p34 were likely to have a poor prognosis, suggesting that this marker may have clinical relevance.

Hypermethylation of O6-methylguanine-DNA methyltransferase promoter may predict nonrecurrence after chemotherapy in colorectal cancer cases.

PURPOSE: Because O(6)-methylguanine-DNA methyltransferase (MGMT) plays an essential role in repairing DNA damage caused by environmental alkylating chemicals, we were interested in determining whether we could see any obvious changes in the properties of colorectal cancers (CRCs) in which the MGMT gene had been silenced by hypermethylation and hence in which very few MGMT protein molecules were being produced. EXPERIMENTAL DESIGN: We used a methylation-specific PCR assay to determine the methylation status of the MGMT promoter in the DNA molecules extracted from CRC and nontumor tissue samples from 116 patients who had undergone CRC surgery and for whom clinical outcome information was available on file. RESULTS: We found evidence of MGMT promoter hypermethylation in 26 of 90 CRC cases, and we noted that the later the stage at which a tumor was diagnosed, the less likely its MGMT promoter was to be methylated (P = 0.03, adjusting for chemotherapy), especially for stage D patients (P = 0.01). We also found that CRC patients with unmethylated MGMT promoters were much more likely to experience recurrence within 36 months than patients with hypermethylated MGMT promoters (crude odds ratio, 14.0; 95% confidence interval, 2.42-81.01). After adjustment for stage, CRC patients with unmethylated MGMT promoters who had been exposed to chemotherapy were found to have a 5.3-fold greater risk of recurrence than those who had no exposure to chemotherapy (95% confidence interval, 1.15-30.92). CONCLUSIONS: Hypermethylation of the MGMT promoter may be predictive of a low risk of recurrence in CRC patients receiving adjuvant chemotherapy.

Long-lasting changes in the T-cell receptor V beta repertoires of CD4 memory T-cell populations in the peripheral blood of radiation-exposed people.

To study the long-term effects of radiation-induced T-cell depletion on the T-cell receptor (TCR) Vbeta repertoires of human peripheral CD4 T-cell populations, we measured the percentages of CD4 T cells representing each of the full range of possible TCR Vbeta families in a cohort of atomic bomb survivors. We then estimated the extent to which the expression levels for individual TCR Vbeta families differed from the average expression level for that particular TCR Vbeta family across the entire cohort. We found no evidence of a systematic change in the TCR Vbeta repertoires of the naïve CD4 T-cell populations, but memory CD4 T-cell TCR Vbeta family expression levels diverged significantly from the population average for counterpart families, especially in individuals who had been exposed to higher doses and were at least 20 years of age at the time of the bombing. Comparisons of the TCR Vbeta family expression profiles in the naïve and memory CD4 T-cell pools of the same group of adult survivors revealed that differences in the TCR Vbeta repertoires of these two types of CD4 T-cell pool were larger in more heavily exposed survivors than in unexposed controls. These findings suggest that the memory CD4 T-cell pools of individuals who received significant radiation doses in adulthood may well have become (and could still be) dependent upon a much less diverse complement of TCR Vbeta families than would otherwise have been the case.

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer naïve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (naïve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (naïve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their naïve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.